KIR engagement prevents Vav tyrosine phosphorylation and the reorganization of actin cytoskeleton.

(A) Tyrosine phosphorylated protein patterns were analyzed by immunoblot from total cell lysates of nonstimulated 4D4 CTLs (lane 1) or incubated 3 minutes at 37°C with RCC-7 (lane 2) or RCC-6 cell lines (lane 3). Ptyr-protein profile of RCC-7 (lane 4) and RCC-6 (lane 5) alone are shown. The lower panel shows membrane reprobing with mAb against SLP-76; * indicates SLP-76 band, the upper band is nonspecific detection of tumor cell proteins. (B) Postnuclear lysates of nonstimulated CTLs (lane 1) or stimulated by RCC-7 targets were immunoprecipitated with polyclonal anti-Vav antibodies and analyzed for tyrosine phosphorylation. CTL was stimulated by RCC-7 alone (lane 2), or in presence of anti–HLA-B/C (lane 3) or control anti–HLA-DR mAbs (lane 4); alternatively CTL was stimulated by RCC-6 (lane 5). Lower panels show membrane reprobing with anti-Vav30 mAb. (C) Confocal analysis was performed as indicated above. 4D4 CTL clone labeled by FITC–CT-B (green) was stimulated with RCC-6 (upper panels) or RCC-7 (lower panels) for 10 minutes at 37°C. After permeabilization, fixed cells were stained with Phalloidin-Texas red (red). This picture represents an overlay and the side-side picture of the overlay showing labeled lipid rafts (FITC–CT-B) or Phalloidin-Texas red as indicated.